Figure 3

Minodronate and alendronate inhibited osteoclast formation via suppression of GGPP biosynthesis in C7 cells. C7 cells were pretreated with 20 μM FOH or 20 μM GGOH for 4 h and were then treated with 0.5 μM minodronate or 2 μM alendronate and 25 ng/mL RANKL plus 50 ng/mL M-CSF for 12 days. Cultures were fed every 3 days by replacing with 500 μL of fresh medium, with or without minodronate, alendronate, RANKL, M-CSF, FOH, and GGOH. (A, B) Inhibitory effect of mevalonate pathway intermediates FOH or GGOH on the inhibition of osteoclast formation by minodronate (A) and alendronate (B). Cultures were fixed and stained for TRAP-positive multinucleated cells, and the number of cells per well was counted. These results are representative of 5 independent experiments. *P < 0.01 compared to 25 ng/mL RANKL plus 50 ng/mL M-CSF administration. (C) CTR and cathepisin K mRNA expression in C7 cells that were treated with minodronate or alendronate along with FOH or GGOH. Total RNA was extracted, and the CTR and cathepsin K mRNA levels were determined by real-time PCR. The results are expressed as the ratio of treated to control samples after normalization to GAPDH mRNA levels. The results are representative of 4 independent experiments. *P < 0.01 compared to 25 ng/mL RANKL plus 50 ng/mL M-CSF administration. (D, E) Cultures were fixed and stained for TRAP-positive cells, and the number of cells per well was counted. These results are representative of 4 independent experiments. *P < 0.01 compared to 25 ng/mL RANKL plus 50 ng/mL M-CSF administration.